Study On The Main Material Basis And Mechanism Of Jing Zhui Huo Xue Capsules In The Treatment Of Ischemic Stroke

Objective:Ischemic stroke refers to the blockage of blood flow in the cerebral arteries and the occurrence of local ischaemia and hypoxia in the brain tissue resulting in impaired neurological function.Jingzhui Huoxue capsules have the therapeutic effect of activating blood circulation,dispersing blood stasis and dispersing cold,and relieving pain.At present,the main material basis and mechanism of action of Jingzhui Huoxue capsules in the treatment of ischaemic stroke are not well defined.In this study,ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS~E)was used to analyse the in vivo and ex vivo components of Jingzhui Huoxue capsules,followed by network pharmacology to screen the main components,action targets and related pathways of Jingzhui Huoxue capsules for the treatment of ischaemic stroke and to investigate the material basis and mechanism of action of their therapeutic effects.Methods:1.In vitro analysis of the chemical composition of Jingzhui Huoxue capsules using UPLC-Q-TOF-MS~E;2.Analysis of blood entry prototypes and their metabolites in model rats orally administered with Jingzhui Huoxue capsules using UPLC-Q-TOF-MS~E;3.Network pharmacology target prediction of in vivo prototype components to screen relevant pathways of Jingzhui Huoxue capsules for the treatment of ischemic stroke;4.PC12 neuronal cells,a oxygen-glucose deprivation/reoxygenation model was established and the PI3K/AKT/VEGFA pathway screened by network pharmacology was validated by MTT,Elisa,RT-q PCR,Western Blot experiments.Results:1.A total of 88 components of Jingzhui Huoxue capsules were identified,mainly flavonoid and its glycosides(12),organic acids(12),saponins(16),volatile oils(27),esters(8)and other components(13);2.12 prototype components and 21 metabolites were identified in a rat model of ischemic stroke among them,flavonoid and its glycosides,saponins and volatile oils may be the main compounds for the efficacy of Jingzhui Huoxue capsules;3.Using network pharmacology methods to predict the target and perform GO and KEGG analysis to construct PPI networks,and using molecular docking techniques to dock the core target and the main active ingredients of the drug to validate and screen the PI3K/AKT/VEGFA signalling pathway;4.(1)The MTT results showed that the optimal concentration of the drug-containing serum in the Jingzhui Huoxue capsules was 10%;(2)From the results of the Elisa experiment,it can be seen that TNF-αexpression was significantly increased(P<0.01)and VEGFA expression was significantly decreased(P<0.01)in the model group compared to the normal group;Compared with the model group,TNF-αexpression was significantly decreased(P<0.01)and VEGFA expression was significantly increased(P<0.05)in the model+drug-containing serum group;(3)RT-q PCR results showed that PI3K,AKT and VEGFA m RNA expression was significantly decreased(P<0.01)and TNF-αm RNA expression was significantly increased(P<0.01)in the model group compared to the normal group;compared with the model group,PI3K,AKT and VEGFA m RNA expression was significantly increased(P<0.01)and TNF-αm RNA expression was significantly decreased(P<0.01)in the model+drug-containing serum group;(4)From the results of Western Blot experiments,it can be seen that the expression of VEGFA,P-PI3K and P-AKT proteins were significantly decreased(P<0.01),TNF-αprotein expression was significantly increased(P<0.01),and PI3K and AKT protein expression,although trending,were not significant(P>0.05)in the model group compared with the normal group;compared with the model group,VEGFA,P-PI3K and P-AKT protein expressions were significantly increased(P<0.05,P<0.01),TNF-αprotein expression was significantly decreased(P<0.01)and PI3K and AKT protein expressions were trending but not significant(P>0.05)in the model+drug-containing serum group。Conclusion:This study combined UPLC-Q-TOF-MS~E with UNIFI software to rapidly and systematically identify the in vivo and ex vivo chemical components of Jingzhui Huoxue capsules;use network pharmacology to predict and analyze the target targets;use PC12cells to validate the PI3K/AKT/VEGFA pathway,and initially elucidate the main material basis and mechanism of action of Jingzhui Huoxue capsules on ischemic stroke.